In eukaryotes including mammals and plants, cytosine methylation and associated heterochromatin formation play a key role in regulating gene expression and genome integrity. Dysregulation of cytosine methylation contributes to human cancers and a number of genetic disorders. Cytosine methylation is also critical for the suppression of transposon expression and movement. We use a group of duplicated methylated endogenous genes in the laboratory plant Arabidopsis as a model system to study the establishment and maintenance of cytosine methylation and gene silencing. In this system, the methylation imprint is established by a double-stranded RNA (dsRNA)-based signal. This mode of methylation targeting was first elucidated in plants, but it is now clear that related mechanisms for RNA-directed heterochromatin formation occur in fungi and animals including mammals. As one aim of this proposal, we will further characterize the RNA signal for DMA methylation using a combination of genetic studies with RNA processing/binding mutants and molecular analysis of RNA, with a focus on understanding the role of short interfering RNAs (siRNAs) in guiding methylation patterning. As a second aim, we will use a novel genetic screen for factors that specifically maintain methylation at a dsRNA source locus. We have also determined that key factors for maintaining the RNA-directed DNA methylation imprint are a cytosine methyltransferase CMT3 and a histone H3 lysine 9 (K9) methyltransferase SUVH4/KYP. This relationship between cytosine methylation and histone H3 K9 methylation also occurs in fungal and mammlian systems. In the third aim of this proposal, we will characterize the stucture and function of SUVH4/KYP and partially redundant related SUVH proteins using biochemical, genetic, and transgenic approaches, with the long-term goal of understanding the connections between RNA, histone methylation, and DNA methylation. [unreadable] [unreadable] [unreadable]